HP1gamma (Phospho-Ser93) Colorimetric Cell-Based ELISA Kit

Product Name:	HP1gamma (Phospho-Ser93)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB01665
ELISA Type:	Cell-Based
Target:	HP1gamma (Phospho-Ser93)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The HP1gamma (Phospho-Ser93) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect HP1gamma protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated HP1gamma in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on HP1gamma phosphorylation.

Qualitative determination of HP1gamma (Phospho-Ser93) concentration is achieved by an indirect ELISA format. In essence, HP1gamma (Phospho-Ser93) is captured by HP1gamma (Phospho-Ser93)-specific primary (1Ãƒ¸) antibodies while the HRP-conjugated secondary (2Ãƒ¸) antibodies bind the Fc region of the 1Ãƒ¸ antibody. Through this binding, the HRP enzyme conjugated to the 2Ãƒ¸ antibody can catalyze a colorimetric reaction upon substrate addition. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:

1.	A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values.
2.	Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method may be used to determine cell density. After staining, the results can be analysed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted.

Database Information:	Gene ID: 11335/653972, UniProt ID: Q13185, OMIM: 604477, Unigene: Hs.381189
Gene Symbol:	CBX3
Sub Type:	Phospho

UniProt Protein Function:	CBX3: Seems to be involved in transcriptional silencing in heterochromatin-like complexes. Recognizes and binds histone H3 tails methylated at 'Lys-9', leading to epigenetic repression. May contribute to the association of the heterochromatin with the inner nuclear membrane through its interaction with lamin B receptor (LBR). Involved in the formation of functional kinetochore through interaction with MIS12 complex proteins. Binds directly to CHAF1A. Interacts with histone H3 methylated at 'Lys-9'. Part of the E2F6.com-1 complex in G0 phase composed of E2F6, MGA, MAX, TFDP1, CBX3, BAT8, EUHMTASE1, RING1, RNF2, MBLR, L3MBTL2 and YAF2. Interacts with LBR, INCENP, TRIM28/TIF1B, SUV420H1, SUV420H2 and SP100. Interacts with TIF1A. Interacts with MIS12 and DSN1. Can interact directly with CBX5 via the chromoshadow domain. Interacts with POGZ. Interacts with CHAMP1. Interacts with ASXL1.
UniProt Protein Details:	Protein type:DNA-binding Chromosomal Location of Human Ortholog: 7p15.2 Cellular Component: chromatin; chromocenter; chromosome, pericentric region; nuclear chromosome, telomeric region; nuclear envelope; nuclear heterochromatin; nuclear inner membrane; nucleus; spindle Molecular Function:enzyme binding; identical protein binding; protein binding; protein domain specific binding Biological Process: chromatin remodeling; negative regulation of transcription, DNA-dependent; rhythmic process; transcription, DNA-dependent
NCBI Summary:	At the nuclear envelope, the nuclear lamina and heterochromatin are adjacent to the inner nuclear membrane. The protein encoded by this gene binds DNA and is a component of heterochromatin. This protein also can bind lamin B receptor, an integral membrane protein found in the inner nuclear membrane. The dual binding functions of the encoded protein may explain the association of heterochromatin with the inner nuclear membrane. This protein binds histone H3 tails methylated at Lys-9 sites. This protein is also recruited to sites of ultraviolet-induced DNA damage and double-strand breaks. Two transcript variants encoding the same protein but differing in the 5' UTR, have been found for this gene.[provided by RefSeq, Mar 2011]
UniProt Code:	Q13185
NCBI GenInfo Identifier:	116241284
NCBI Gene ID:	11335
NCBI Accession:	Q13185.4
UniProt Secondary Accession:	Q13185,Q96CD7, Q99409, Q9BVS3, Q9P0Z6,
UniProt Related Accession:	Q13185
Molecular Weight:	20,811 Da
NCBI Full Name:	Chromobox protein homolog 3
NCBI Synonym Full Names:	chromobox 3
NCBI Official Symbol:	CBX3
NCBI Official Synonym Symbols:	HECH; HP1-GAMMA; HP1Hs-gamma
NCBI Protein Information:	chromobox protein homolog 3
UniProt Protein Name:	Chromobox protein homolog 3
UniProt Synonym Protein Names:	HECH; Heterochromatin protein 1 homolog gamma; HP1 gamma; Modifier 2 protein
Protein Family:	Chromobox protein
UniProt Gene Name:	CBX3
UniProt Entry Name:	CBX3_HUMAN

Component	Quantity
96-Well Cell Culture Clear-Bottom Microplate	2 plates
10X TBS	24 mL
Quenching Buffer	24 mL
Blocking Buffer	50 mL
15X Wash Buffer	50 mL
Primary Antibody Diluent	12 mL
100x Anti-Phospho Target Antibody	60 µL
100x Anti-Target Antibody	60 µL
Anti-GAPDH Antibody	60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody	12 mL
HRP-Conjugated Anti-Mouse IgG Antibody	12 mL
SDS Solution	12 mL
Stop Solution	24 mL
Ready-to-Use Substrate	12 mL
Crystal Violet Solution	12 mL
Adhesive Plate Seals	2 seals

The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:

Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional)
Micropipettes with capability of measuring volumes ranging from 1 µL to 1 ml
37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
Graph paper or computer software capable of generating or displaying logarithmic functions
Absorbent papers or vacuum aspirator
Test tubes or microfuge tubes capable of storing ≥1 ml
Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
Orbital shaker (optional)
Deionized or sterile water

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Step	Procedure
1.	Seed 200 µL of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µL of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37 °C prior to adding cells.
2.	Incubate the cells for overnight at 37 °C, 5% CO2.
3.	Treat the cells as desired.
4.	Remove the cell culture medium and rinse with 200 µL of 1x TBS, twice.
5.	Fix the cells by incubating with 100 µL of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells.
6.	Remove the Fixing Solution and wash the plate 3 times with 200 µL 1x Wash Buffer for five minutes each time with gentle shaking on the orbital shaker. The plate can be stored at 4 °C for a week.
7.	Add 100 µL of Quenching Buffer and incubate for 20 minutes at room temperature.
8.	Wash the plate 3 times with 1x Wash Buffer for 5 minutes each time.
9.	Add 200 µL of Blocking Buffer and incubate for 1 hour at room temperature.
10.	Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
11.	Add 50 µL of 1x primary antibodies Anti-HP1gamma (Phospho-Ser93) Antibody, Anti-HP1gamma Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover with Parafilm and incubate for 16 hours (overnight) at 4 °C. If the target expression is known to be high, incubate for 2 hours at room temperature.
12.	Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
13.	Add 50 µL of 1x secondary antibodies (HRP-Conjugated AntiRabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature.
14.	Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
15.	Add 50 µL of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark.
16.	Add 50 µL of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader.

(Additional Crystal Violet staining may be performed if desired – details of this may be found in the kit technical manual.)